Extracellular nucleotides show pro-survival or detrimental effects on the CNS under acute or chronic conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by various ATP metabolizing enzymes. We are interested in verifying if and how purinergic signalling can contribute to the survival, repair and regeneration of neurons following oxygen-glucose deprivation (OGD), which mimics in vitro the cytotoxicity induced by ischemia in vivo. Our experimental model consist on organotypic co-cultures of rodent brain slices from mesencephalic ventral tegmental area/substantia nigra (VTA-SN) and prefrontal cortex (PFC), maintained in culture up to 3 weeks, to study the effect of purinergic agents on nerve fibers regeneration and on the reconstruction of neuronal circuitries under control conditions and after OGD. Cytotoxicity assay showed significant tissue damage after 1h OGD and this data correlated with the increased number of PI-positive cells in OGD cultures. We have then focussed on the effects on neurons and on the glial population in order to understand the modification of the glial response after injury and its possible role in damage propagation or recovery: preliminary data suggest that in the PFC the number of astroglial cells is not affected by OGD, while an induction of microglial cells activation and an increased number of oligodendroglial cells were detected. Because the activity of some nucleotide-hydrolysing enzymes have been reported to increase upon ischemia, we have set up a lead-phosphate method for the in situ localization of their activity, characterized and validated in dissociated cell cultures from rat CNS. Results highlight that the exposure of these cells to hypoxic/hypoglycaemic conditions results in an increased activity of ATP-hydrolyzing enzymes. We are currently translating this method to the in vitro organotypic VTA-SN/PFC cultures.
Role of ATP and ATP-metabolizing enzymes in neuroreparative processes after hypoxic-ischemic injury in rodent organotypic brain slices / L. Colombo, S.M. Ceruti, G. Magni, M. Boccazzi, E. Dossi, W. Wanke, P. Illes, M.P. Abbracchio. ((Intervento presentato al convegno Next Step : la giovane ricerca avanza tenutosi a Milano nel 2010.
Role of ATP and ATP-metabolizing enzymes in neuroreparative processes after hypoxic-ischemic injury in rodent organotypic brain slices
L. Colombo;S.M. Ceruti;G. Magni;M. Boccazzi;M.P. Abbracchio
2010
Abstract
Extracellular nucleotides show pro-survival or detrimental effects on the CNS under acute or chronic conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by various ATP metabolizing enzymes. We are interested in verifying if and how purinergic signalling can contribute to the survival, repair and regeneration of neurons following oxygen-glucose deprivation (OGD), which mimics in vitro the cytotoxicity induced by ischemia in vivo. Our experimental model consist on organotypic co-cultures of rodent brain slices from mesencephalic ventral tegmental area/substantia nigra (VTA-SN) and prefrontal cortex (PFC), maintained in culture up to 3 weeks, to study the effect of purinergic agents on nerve fibers regeneration and on the reconstruction of neuronal circuitries under control conditions and after OGD. Cytotoxicity assay showed significant tissue damage after 1h OGD and this data correlated with the increased number of PI-positive cells in OGD cultures. We have then focussed on the effects on neurons and on the glial population in order to understand the modification of the glial response after injury and its possible role in damage propagation or recovery: preliminary data suggest that in the PFC the number of astroglial cells is not affected by OGD, while an induction of microglial cells activation and an increased number of oligodendroglial cells were detected. Because the activity of some nucleotide-hydrolysing enzymes have been reported to increase upon ischemia, we have set up a lead-phosphate method for the in situ localization of their activity, characterized and validated in dissociated cell cultures from rat CNS. Results highlight that the exposure of these cells to hypoxic/hypoglycaemic conditions results in an increased activity of ATP-hydrolyzing enzymes. We are currently translating this method to the in vitro organotypic VTA-SN/PFC cultures.
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